Cloning and expression of thermostable β-amylase gene of Thermoanaerobacterium thermosulfurogenes in Escherichia coli and Bacillus subtilis BR151

Thermoanaerobacterium thermosulfurogenes DNA’sından sıcaklığa dirençli β-amilaz genini kodlayan DNA parçası PCR ile amplifiye edilerek pBluescript II KS/SK, pBT10, pNW33N ve pUB110 plazmidlerine klonlanmıştır. Rekombinant plazmidler sırasıyla pBluescriptβ, pBT10β, pNW33Nβ ve pUB110β olarak isimlendirilmişlerdir. pBluescriptβ, pBT10β ve pNW33Nβ rekombinant plazmidleri Escherichia coli, pUB110β plazmidi ise Bacillus subtilis BR151 bakterisine transfer edilmişlerdir. E. coli ve B. subtilis bakterilerinden izole edilen rekombinant plazmidlerin insört ve PCR analizleri, agaroz jel elektroforezde 1935 bç’lik β-amilaz genini doğrulamıştır. LB-nişasta-agar plaklarında, tüm rekombinant E. coli kolonileri I2 boyaması ile pozitif zon vermişlerdir. Sıcaklığa dirençli β-amilaz geni B. subtilis BR151 bakterisinde klonlanmasına karşın, LB-nişasta-agar plağında enzimatik aktivite gözlenememiştir. B. subtilis kökenli hücre dışı proteinlerin renatürasyonundan sonra, SDS-Nişasta-PAGE’de β-amilaz enzimatik aktivitesini tekrar kazanmış ve bu sonuç B. subtilis konukçusunda enzimin uygun katlanmayı yapamadığı görüşünü desteklemiştir.

Anahtar Kelimeler:

elektroforez, Bacillus subtilis, DNA, DNA klonlama, Escherichia coli, bakteriyel proteinler, beta-amilaz, enzim aktivitesi, enzimler, gen ifadesi, genler, plazmitler, polimeraz zincir reaksiyonu

Thermoanaerobacterium thermosulfurogenes’e ait sıcaklığa dirençli β-amilaz geninin Escherichia coli ve Bacillus subtilis BR151’de klonlanması ve ekspresyonu

DNA fragment encoding thermostable β-amylase gene from Thermoanaerobacterium thermosulfurogenes was amplified by PCR and then cloned into pBluescript II KS/SK, pBT10, pNW33N and pUB110 plasmids. Recombinant plasmids were designated as pBluescriptβ, pBT10β, pNW33Nβ and pUB110β respectively. pBluescriptβ, pBT10β and pNW33Nβ recombinant plasmids were transferred into Escherichia coli, and pUB110β was electrotransformed into Bacillus subtilis BR151. Insert and PCR analysis of recombinant plasmids from E. coli and B. subtilis confirmed the 1935 bp β-amylase gene fragment on agarose gel electrophoresis. On LB-starch-agar plates, all recombinant E. coli colonies showed positive zones with I2 staining. Although thermostable β-amylase gene was cloned in B. subtilis BR151, the enzyme activity was not detected on LB-starch-agar plate. But after renaturation of extracellular proteins from B. subtilis on SDS-Starch-PAGE, β-amylase enzyme regained enzymatic activity by zymogram technique and thereby confirmed that enzyme was not folding properly in B. subtilis host.

Keywords:

electrophoresis, Bacillus subtilis, DNA, DNA cloning, Escherichia coli, bacterial proteins, beta-amylase, enzyme activity, enzymes, gene expression, genes, plasmids, polymerase chain reaction,

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